A. HEMATOPOIESIS
The process of blood cell formation whereby red and white blood cells are replaced through the division of cells located in the bone marrow is called hematopoiesis. For a review of hematopoiesis see Dexter and Spooncer (Ann. Rev. Cell Biol., 3:423-441 [1987]).
There are many different types of blood cells which belong to distinct cell lineages. Along each lineage, there are cells at different stages of maturation. Mature blood cells are specialized for different functions. For example, erythrocytes are involved in O.sub.2 and CO.sub.2 transport; T and B lymphocytes are involved in cell and antibody mediated immune responses, respectively; platelets are required for blood clotting; and the granulocytes and macrophages act as general scavengers and accessory cells. Granulocytes can be further divided into basophils, eosinophils, neutrophils and mast cells.
Each of the various blood cell types arises from pluripotent or totipotent stem cells which are able to undergo self-renewal or give rise to progenitor cells or Colony Forming Units (CFU) that yield a more limited array of cell types. As stem cells progressively lose their ability to self-renew, they become increasingly lineage restricted. It has been shown that stem cells can develop into multipotent cells (called "CFC-Mix" by Dexter and Spooncer, supra). Some of the CFC-Mix cells can undergo renewal whereas others lead to lineage-restricted progenitors which eventually develop into mature myeloid cells (e.g., neutrophils, megakaryocytes, macrophages, basophils and erythroid cells). Similarly, pluripotent stem cells are able to give rise to PreB and PreT lymphoid cell lineages which differentiate into mature B and T lymphocytes, respectively. Progenitors are defined by their progeny, e.g., granulocytelmacrophagecolony-forming progenitor cells (GM-CFU) differentiate into neutrophils or macrophages; primitive erythroid burst-forming units (BFU-E) differentiate into erythroid colony-forming units (CFU-E) which give rise to mature erythrocytes. Similarly, the Meg-CFU, Eos-CFU and Bas-CFU progenitors are able to differentiate into megakaryocytes, eosinophils and basophils, respectively.
The number of pluripotent stem cells in the bone marrow is extremely low and has been estimated to be in the order of about one per 10,000 to one per 100,000 cells (Boggs et al., J. Clin. Inv., 70:242 [1982] and Harrison et al., PNAS, 85; 822 [1988]). Accordingly, characterization of stem cells has been difficult. Therefore, various protocols for enriching pluripotent stem cells have been developed. See, for example, Matthews et al., Cell, 65:1143-1152 [1991]; WO 94/02157; Orlic et al., Blood, 82(3 :762-770 [1993]; and Visseret al., Stem Cells, 11Suppl. (2):49-55 [July 1993].
Various lineage-specific factors have been demonstrated to control cell growth, differentiation and the functioning of hematopoietic cells. These factors or cytokines include the interleukins (e.g., IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (M-CSF), erythropoietin (Epo), Iymphotoxin, steel factor (SLF), tumor necrosis factor (TNF) and gamma-interferon. These growth factors have a broad spectrum of activity, from generalized to lineage-specific roles in hematopoiesis, or a combination of both. For example, IL-3 appears to act on multipotent stem cells as well as progenitors restricted to the granulocytelmacrcphage, eosinophil, megakaryocyte, erythroid or mast cell lineages. On the other hand, Epo generally acts on fairly mature erythroid progenitor cells.
B. THE HEMATOPOIETIC ENVIRONMENT AND EMBRYOGENESIS
The capacity of the hematopoietic stem cells to provide for the lifelong production of all blood lineages is accomplished by a balance between the plasticity of the stem cell, that is the production of committed progenitors cells which generate specific blood lineages, and the replication of the stem cell in the undifferentiated state (self-renewal). The mechanisms regulating hematopoietic stem cells' plasticity and self-renewal in vivo have been difficult to define. However, the major contributory factors represent a combination of cell intrinsic and environmental influences (Morrison et al., Proc. Natl. Acad. Sci. USA, 92: 10302-10306 [1995]). The importance of the hematopoieticmicroenvironmenthas been established through the use of long term bone marrow culture systems where hematopoieticcells cultured on stroma allow for the maintenance of HSCs, albeit at low frequencies (Fraser et al., Proc. Natl. Acad. Sci. USA, 89: 1968-1972, [1992]; Wineman et al., Blood, 81: 365-372 [1993]).
The demonstration of hematopoietic cell maintenance in culture has led to efforts to identify candidate `stem cell` factors. The role of hematopoietic cytokines in stem cell maintenance has been studied by direct addition of purified factors to in vitro cultures of stem cell populations followed by transplantation of the cultured cells (Muench et al., Blood, 81: 3463-3473 [1993]; Wineman et al., supra [1993]; Rebel et al., Blood, 83: 128-136 [1994]). Most of the known `early-acting` cytokines such as IL-3, IL-6, and KL have been shown to stimulate proliferation of more committed progenitor cells while concurrently allowing maintenance, but not expansion, of cells capable of long-term multilineage repopulation (reviewed in Williams, Blood, 81(12):3169-3172 [1993]; Muller-Sieburg and Deryugina, Stem Cells, 13: 477-486 [1995]). While these data indicate that the cells' plasticity and repopulating function may be preserved by cytokine treatment, the molecules that promote self-renewal of these pluripotent cells remain unknown.
Transplantation studies have shown that the signals that regulate fate pluripotent stem cells may be similar in the embryo and adult bone marrow. Cells from the day 11 fetal liver, yolk sac, or aorta/gonad/mesonephros (AGM) region can repopulate the adult marrow and appropriately respond to extrinsic cues to sustain long-term multilineage hematopoiesis (Muller et al., Immunity, 1:291-301 [1994]). Although embryonic hematopoiesis is largely devoted to the erythroid lineage, the embryonic microenvironment clearly contributes to the maintenance of pluripotent stem cells in the undifferentiatedstate. These cell populations are cycling during embryogenesis (Zeigler et al., supra [1994]; Morrison et al., supra [1995]; Rebel et al., Blood, 87: 3500-3507 [1996]).
In mammals, hematopoietic precursors arise in the extraembryonic and ventral mesoderm, yolk sac, or AGM region (Dzierzakand Medvinsky Trends Genet., 11: 359-366 [1995]; Zon, Blood, 86: 2876-2891 [1995]). In amphibian embryos, the equivalent regions are the ventral blood island mesoderm and the dorsal lateral plate mesoderm (reviewed in Kessler and Melton, Science, 266: 596-604 [1994]; Zon, supra 1995; Tam and Quinlan, Curr. Biol., 6: 104-106 [1996]). Secreted factors that potentially regulate cell fate determination of ventral mesoderm in Xenopus include Wnts, FGFs, and BMP-4 (reviewed in Christian and Moon, Bio Essays, 15: 135-140 [1993a]; Zon, supra [1995]). Embryonic expression of XWnt-8 (Christian and Moon, Genes Dev., 7: 13-28 [1993b]) and XWnt-11(Ku and Melton, Development, 119: 1161-1173 1993]) is localized to the area of prospective ventral and lateral mesoderm and XWnt-8 expression can be induced by ventralizing factors such as FGFs and BMP-4.
C. THE WNTS GENE FAMILY
Wnts are encoded by a large gene family whose members have been found in round worms, insects, cartilaginous fish and vertebrates (Sidow, 1994). Wnts are thought to function in a variety of developmental and physiological processes since many diverse species have multiple conserved Wnt genes (McMahon, Trends Genet., 8: 236-242 [1992]; Nusse and Varmus, Cell, 69: 1073-1087 [1992]). Wnt genes encode secreted glycoproteins that are thought to function as paracrine or autocrine signals active in several primitive cell types (McMahon, supra [1 992]; Nusse and Varmus, supra [1992]). The Wnt growth factor family includes more than 10 genes identified in the mouse (Wnt-1, 2, 3a, 3b, 4, 5a, 5b, 6, 7a 7b, 8a, 8b, 10b, 11, 12) (see, e.g., Gavin et al., Genes Dev., 4: 2319-2332 [1990]; Lee et al., Proc. Natl. Acad. Sci. USA, 92: 2268-2272; Christiansen et al., Mech. Dev. 51: 341-350 [1995]) and at least 7 genes identified in the human (Wnt-1, 2, 3,4, 5a, 7a and 7b) by cDNA cloning (see, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534 [1984]). The Wnt-1 proto-oncogene (int-1) was originally identified from mammary tumors induced by mouse mammary tumor virus (MMTV) due to an insertion of viral DNA sequence (Nusse and Varmus, Cell, 31: 99-109 [1982]). In adult mice, the expression level of Wnt-1 mRNA is detected only in the testis during later stages of sperm development. Wnt-1 protein is about 42 KDa and contains an amino terminal hydrophobic region, which may function as a signal sequence for secretion (Nusse and Varmus, supra). The expression of Wnt-2/irp is detected in mouse fetal and adult tissues and its distribution does not overlap with the expression pattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis. The expression of Wnt-3 in mouse embryos detected in the neural tubes and in the limb buds. Wnt-5a transcripts are detected in the developing fore- and hind limbs at 9.5 through 14.5 days and highest levels are concentrated in apical ectoderm at the distal tip of limbs (Nusse and Varmus, supra [1992]. Recently, a Wnt growth factor, termed Wnt-x, was described (PCT/US94/14708; W095/17416) along with the detection of Wnt-x expression in bone tissues and in bone-derived cells. Also described was the role of Wnt-x in the maintenance of mature osteoblasts and the use of the Wnt-x growth factor as a therapeutic agent or in the development of other therapeutic agents to treat bone-related diseases.
Wnts may play a role in local cell signaling. Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freely diffusible in the medium (Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730 [1990]; Bradley and Brown, EMBO J., 9: 1569-1575 [1990]).
Studies of mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning. In Drosophila, wingless (wg) encodes a Wnt gene (Rijsenijk et al., Cell. 50: 649-657 [1987]) and wg mutations alter the pattern of embryonic ectoderm, neurogenesis, and imaginal disc outgrowth (Morata and Lawrence, Dev. Biol., 56: 227-240 [1977]; Baker, Dev. Biol., 125: 96-108 [1988]; Klingensmith and Nusse, Dev. Biol., 166: 396-414[1994]). In Caenorhabditis elegans, lin-44 encodes a Wnt which is required for asymmetric cell divisions (Herman and Horvitz, Development, 120: 1035-1047 [1994]). Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradley, Cell, 62: 1073-1085 [1990]; Thomas and Cappechi, Nature, 346: 847-850 [1990]), and the outgrowth of embryonic primordia for kidney (Stark et al., Nature, 372: 679-683 [1994]), tail bud (Takada et al., Genes Dev., 8: 174-189 [1994]), and limb bud (Parr and McMahon, Nature, 374: 350-353 [1995]). Overexpression of Wnts in the mammary gland can result in mammary hyperplasia (McMahon, supra (1992]; Nusse and Varmus, supra [1992]), and precocious alveolar development (Bradbury et al., Dev. Biol., 170: 553-563 [1995]). A role for Wnts in mammalian hematopoiesis has not previously been suggested or considered.
Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesoderm and the extraembryonic mesoderm of the day 7-8 murine embryo (Gavin et al., supra [1990]). These embryonic domains contribute to the AGM region and yolk sac tissues from which multipotent hematopoietic precursors and HSCs are derived (Dzierzak and Medvinsky, supra [1995]; Zon, supra [1995], Kanatsu and Nishikawa, Development, 122: 823-830 [1996]). Wnt-5a, Wnt-10b, and other Wnts have been detected in limb buds, indicating possible roles in the development and patterning of the early bone microenvironment as shown for Wnt-7b (Gavin et al., supra [1990]; Christiansen et al., Mech. Devel., 51: 341-350 [1995]; Parr and McMahon, supra [1995]).
D. HEMATOPOIETIC DISEASES AND DISORDERS
Chemo- and radiation therapies cause dramatic reductions in blood cell populations in cancer patients. At least 500,000 cancer patients undergo chemotherapy and radiation therapy in the US and Europe each year and another 200,000 in Japan. Bone marrow transplantation therapy of value in aplastic anemia, primary immunodeficiency and acute leukemia (following total body irradiation) is becoming more widely practiced by the medical community. At least 15,000 Americans have bone marrow transplants each year. Other diseases can cause a reduction in entire or selected blood cell lineages. Examples of these conditions include anemia (including macrocytic and aplastic anemia); thrombocytopenia; hypoplasia; immune (autoimmune) thrombocytopenic purpura (ITP); and HIV induced ITP.
Pharmaceutical products are needed which are able to enhance reconstitution of blood cell populations of these patients.
Accordingly, it is an object of the present invention to provide a method for enhancing the proliferation and/or differentiation and/or maintenance of primitive hematopoietic cells. Such a method may be useful for enhancing repopulation of hematopoietic stem cells and thus mature blood cell lineages. This is desirable where a mammal has suffered a decrease in hematopoietic or mature blood cells as a consequence of disease, radiation or chemotherapy. This method is also useful for generating expanded populations of such stem cells and mature blood cell lineages from such hematopoietic cells ex vivo.
These and other objects will be apparent to the ordinary artisan upon consideration of the specification as a whole.